Field of Invention
The present disclosure relates to cell lines and mouse strains, more particularly, to mouse cell line authentication.
Description of Related Art
Cell line authentication is becoming increasingly important. For example, cell line authentication is now required by some journals prior to publication. In some cases, cell line authentication may be mandatory before receiving funding from some granting agencies.
The Federal Drug Administration has also instituted a requirement for the authentication of cell lines used to produce pharmaceuticals. Methods are currently in place for authenticating human cell lines using multiplex PCR assays that target short tandem repeat (STR) markers in the human genome and are capable of generating a unique individual genotypic profile. Cell repositories may now genotype their human cell lines using at least eight human STR markers including a marker for amelogenin, the sex identification locus.
Large databases of STR profiles may be used to confirm genotypes of human cell lines. These databases may also be used to provide a record of previously misidentified or cross-contaminated human cell lines. The American Type Culture Collection (ATCC) website maintains an updated list of misidentified human cell lines. Although there are successful methods in place for human cell line authentication, methods for nonhuman cell lines are not well established.
Mouse cell lines are commonly used to study human genes and disease. Mouse cells may also be used in the biomanufacturing of recombinant proteins and may also function as feeder cells for embryonic stem cells.
In the prior art, there are a number of techniques used to identify mouse cell lines or mouse strains, including but not limited to microsatellite markers (simple sequence length polymorphism (SSLP) or STR markers), single nucleotide polymorphisms (SNPs), and species-specific primers. One of the most commonly used laboratory strains is the Mus musculus domesticus strain. This strain may be difficult to genotype due to many shared alleles as a result of extensive breeding.
There have been published reports of multiplexing mouse SSLPs post-PCR by pooling the amplified products to distinguish between different strains of inbred mice. However, most of the microsatellite markers that have been used for these purposes are dinucleotide in nature, mainly CA repeats, which may result in noisy stutter and have relatively high mutation frequencies when compared to tetranucleotide repeats. The reduced stutter associated with tetranucleotide repeats may allow for easier interpretation of single and mixed profiles.
There is a need for mouse cell line authentication based on tetranucleotide repeats, as opposed to microsatellite markers that are dinucleotide in nature.
Current methods may lack the resolution to differentiate between individual mice of the same subspecies. SNPs may be well-conserved between inbred mice of the same strain. Thus, it may be difficult to differentiate between interstrain mice using this method. Even an extensive array containing over 600,000 mouse SNPs may still be unable to identify individual mice within the same subspecies.
Species-specific primers may be used to determine the origin of species for cell lines. However, species-specific primers may lack specificity to identify at the individual level.
For human cell lines, practices have been adopted that are currently used to identify such cells. These practices are based on the detection of short tandem repeat (STR) markers. However, for the large volume of research that is performed using nonhuman cell lines (mouse, hamster, monkey, etc.), there are no STR marker assays that are able to uniquely and unequivocally identify a particular cell line.
There is further a need for a mouse cell line authentication assay that uniquely identifies cell lines at the individual level.